This invention relates to the selective separation of high density lipoproteins from low density lipoproteins in human serum. Another aspect of this invention relates to the selective precipitation of low density lipoproteins from human serum using a reagent which contains no divalent metal ions. Still another aspect of this invention relates to a method for separating high density lipoproteins from low density lipoproteins in human serum and quantitatively determining the high density lipoprotein components present in human serum.
For many years now, the medical profession has been concerned with the quantity of cholesterol present in human serum. Total serum cholesterol analysis has proven useful in the diagnosis of hyperlipoproteinemia, atherosclerosis, and hepatic and thyroid diseases. In the past, high cholesterol levels in human serum were linked to coronary heart disease and physicians often prescribed drugs to remove or greatly reduce cholesterol serum levels. Cholesterols or lipoproteins can be divided into two classes of related, though not identical, molecules. Although these fractions are not homogenous, their chemical and physical properties and metabolic interrelationships indicate that they do represent distinct classes of related molecules rather than arbitrary fractions defined by methods used in their isolation. Recently, it has been established that an inverse relationship exists between serum high density lipoproteins (high density cholesterol) and the risk of coronary heart disease. It has been further determined that the low density lipoproteins (low density cholesterol) deposit plaque on the inside of arteries whereas the high density lipoproteins actually dissolve plaque on the inside of the arteries. Thus, an accurate, reproducible and dependable in vitro quantitative test is important in diagnostic medicine because of the beneficial effects of high density lipoproteins and the harmful effects of low density lipoproteins. High density lipoprotein analysis is especially important for the assessment of coronary heart disease risk in apparently normal individuals.
In the past, low density lipoproteins have been precipitated with sodium phosphotungstate and magnesium chloride; with sulfated polysaccharides at a neutral pH; with polysaccharides of high molecular weight at a neutral pH; and with lower molecular weight polysaccharides, such as heparin in the presence of divalent cations. These methods are disclosed in Cornwell, D. G., and F. A. Kruger, 1961, J. Lipid Res. 2:10, and Burstein, M., H. R. Scholnick and R. Morfin, 1970, J. Lipid Res. 11:583. Another method used to separate high density lipoproteins is ultracentrifugation which can be slow and cumbersome.
Problems can occur in practicing these methods of separating high density lipoproteins from low density lipoproteins, especially in quantitative determinations. For example, previous quantitative methods have been based on metal polyanion precipitation including such combinations as divalent manganese cations plus heparin, divalent magnesium cations plus sodium phosphotungstate and divalent calcium cations plus dextran sulfate. The presence of metal cations causes interference in the subsequent cholesterol enzymatic assay. Further, higher concentrations of polyanions were previously required which inhibited the enzymatic cholesterol assay, preventing accurate and reproducible determinations of high density lipoprotein components. Also, previous precipitation methods were time and temperature dependent.
Thus, a need exists for a precipitating reagent and method using a low concentration of polyanions without metal ions to avoid inhibition of the cholesterol assay, which is accurate, reproducible and not time or temperature dependent.